A Major Secreted Elastase Is Essential for Pathogenicity of
 Aeromonas hydrophila

Cascón, Alberto; Yugueros, J; Temprano, A; Sánchez, María de la Paz; Hernanz, Carmen; Luengo, José M.; Naharro, Germán

doi:10.1128/iai.68.6.3233-3241.2000

Cited by 139 publications

(111 citation statements)

References 48 publications

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“…14) As described later, the genetic analysis showed that the structure gene of AMP of A. hydrophila 453 is composed of 1767 bp and AMP is synthesized as the molecule consisting of 589 amino acid residues. The theoretical molecular size of the molecule is 62554.5 Da.…”

Section: Resultsmentioning

confidence: 99%

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Analysis of Carboxy Terminal Domain of Metalloprotease of Elastolytic Aeromonas hydrophila

Takahashi

Kobayashi

Yamanaka

et al. 2013

Biological & Pharmaceutical Bulletin

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We examined the ability of Aeromonas hydrophila to lyse elastin. Eight of 13 strains showed elastolytic activity on agar medium containing elastin and 5 strains did not. In order to examine the involvement of the metalloprotease of A. hydrophila (AMP) in elastolytic activity, we made the amp-deletion mutant strain from an elastolytic strain. The elastolytic activity of the strain decreased with this deletion. The analysis of AMP released into the culture supernatant showed that AMP appeared outside of the cell as the intermediate consisting of a mature domain and carboxy terminal (C-terminal) propeptide domain. Further analysis showed that the intermediate has the ability to lyse elastin and that loss of the C-terminal domain causes loss of the elastolytic activity of the intermediate. We then determined the nucleotide sequence of the amps of all strains used in this study. Phylogenetic analysis revealed that these AMPs were divided into three groups. The AMPs from elastolytic strains belong to group I or group II, and AMPs from non-elastolytic strains belong to group III. The distance between group I and group II is small, but group III is located separately from groups I and II. Comparison of the amino acid residues of the C-terminal domain revealed that there are 13 amino acid residues specific to the C-terminal domain of group III. This indicates that the conformation of the C-terminal propeptide domain formed by these specific amino acid residues is important for AMP to express elastolytic activity. Key words Aeromonas hydrophila; metalloprotease; elastolytic activityThe mesophilic Aeromonas species are ubiquitous in aquatic environments.1-4) The mesophilic species cause sporadic diarrhea in both adults and children, 5,6) and it has been reported that A. hydrophila and A. sobria occasionally cause extraintestinal infections such as sepsis and necrotizing softtissue infection in patients. Such extraintestinal infections often occur in patients suffering from hepatic dysfunction and diabetes mellitus. [7][8][9] As pathogenic factors of Aeromonas, various extracellular proteins containing hemolysin, proteases, lipases and so on have been reported. 10,11) In addition, a virulent type III secretion system has been proposed.12) The hemolysin produced by A. hydrophila and A. sobria is thought to be a major etiological agent of diarrhea because it induces fluid accumulation in the intestinal loop test and the antiserum against hemolysin neutralizes the enterotoxic activity of these bacteria 13) ; however, it is unclear how A. hydrophila and A. sobria, which infect the intestine, cause exointestinal infection.Cascón et al. have reported that elastase of A. hydrophila plays an important role in the pathogenesis of the bacteria. They determined the LD 50 of two isogenic strains of A. hydrophila, wild-type strain and the elastase gene-deleted mutant strain of the wild type by intraperitoneal challenge of rainbow trout. The LD 50 of the mutant strain is about 100 times higher than that of the wild type, indicating that ...

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Section: Resultsmentioning

confidence: 99%

“…Furthermore, they demonstrated that the elastase is the metalloprotease of A. hydrophila (AMP) and that it also hydrolyzes casein. 14) In the previous study, we examined the properties of the metalloprotease produced by A. sobria. 15) A. sobria, which was used in the previous experiment, showed caseinolytic activity, but did not show elastolytic activity.…”

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confidence: 99%

Analysis of Carboxy Terminal Domain of Metalloprotease of Elastolytic Aeromonas hydrophila

Takahashi

Kobayashi

Yamanaka

et al. 2013

Biological & Pharmaceutical Bulletin

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“…Several studies concerning the survival of A. hydrophila strains in aquatic environments, such as distilled water (44), mineral water (18,45,47), tap water (43), and seawater (40), have been published. Microcosms are often used in these studies, most of them applied to assess the health impact of the presence of motile Aeromonas species in drinking water, because the production of a wide range of virulence factors is a common feature of virulent strains (10,13,53,54,64).…”

Section: Cfu MLmentioning

confidence: 99%

Behavior of an Aeromonas hydrophila aroA Live Vaccine in Water Microcosms

Vivas¹,

Carracedo²,

Riaño³

et al. 2004

Appl Environ Microbiol

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Genetically modified auxotrophic mutants of different fish pathogens have been used as live vaccines in laboratory experiments, but the behavior of the strains after release into aquatic ecosystems has not been characterized. We previously constructed and characterized an aroA mutant of Aeromonas hydrophila and studied the protection afforded by this mutant as a live vaccine in rainbow trout. In this work, we describe the survival of this strain in aquatic microcosms prepared from fish water tanks. The aroA mutant disappeared rapidly in nonfiltered, nonautoclaved fish tank water, declining below detection levels after 15 days, suggesting an inhibitory effect of the autochthonous microflora of the water. When the aroA strain was used to inoculate sterilized water, its culturability was lower than that of wild-type strain A. hydrophila AG2; after long periods of incubation, aroA cells were able to enter a viable but nonculturable state. Entry into this nonculturable state was accompanied by changes in the cell morphology from rods to spheres, but the cells appeared to remain potentially viable, as assessed by the preservation of cell membrane integrity. Supplementation of the culture medium with sodium pyruvate favored the culturability and resuscitation of the two A. hydrophila strains at low temperatures (6 and 16°C). These results contribute to a better understanding of the behavior of the aroA strain in natural environments and suggest that the inactivation of the aroA gene may be beneficial for the safety of this live vaccine for aquacultures.

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“…Disruption of more than one gene or a whole gene cluster as in the case of TTSS appears to be necessary to observe differences in the LD 50 s. On the other hand, the virulence of A. hydrophila may be also bacterial strain, infection route, and animal model dependent. It has been reported that a single mutation in cytotoxic enterotoxin of A. hydrophila SSU resulted in an ϳ300-fold increase in LD 50 s by intraperitoneal injection in Swiss-Webster mice (44); a single mutation in elastase of A. hydrophila AG2 resulted in an ϳ100-fold increase in LD 50 s by intraperitoneal challenge in rainbow trout (5). In addition, double deletions mutations were made in strain AH-1, such as S-layer and metalloprotease (⌬ahsA ⌬mepA; LD 50 ϭ 10 5.7 ), S-layer and serine protease (⌬ahsA ⌬serA; LD 50 ϭ 10 5.7 ), and a triple-deletion mutant of S-layer, metalloprotease, and serine protease (⌬ahsA ⌬serA ⌬mepA; LD 50 ϭ 10 6.5 ).…”

Section: Resultsmentioning

confidence: 99%

Identification and Characterization of Putative Virulence Genes and Gene Clusters in Aeromonas hydrophila PPD134/91

Zhang

Lau

et al. 2005

Appl Environ Microbiol

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Aeromonas hydrophila is a gram-negative opportunistic pathogen of animals and humans. The pathogenesis of A. hydrophila is multifactorial. Genomic subtraction and markers of genomic islands (GIs) were used to identify putative virulence genes in A. hydrophila PPD134/91. Two rounds of genomic subtraction led to the identification of 22 unique DNA fragments encoding 19 putative virulence factors and seven new open reading frames, which are commonly present in the eight virulence strains examined. In addition, four GIs were found, including O-antigen, capsule, phage-associated, and type III secretion system (TTSS) gene clusters. These putative virulence genes and gene clusters were positioned on a physical map of A. hydrophila PPD134/91 to determine their genetic organization in this bacterium. Further in vivo study of insertion and deletion mutants showed that the TTSS may be one of the important virulence factors in A. hydrophila pathogenesis. Furthermore, deletions of multiple virulence factors such as S-layer, serine protease, and metalloprotease also increased the 50% lethal dose to the same level as the TTSS mutation (about 1 log) in a blue gourami infection model. This observation sheds light on the multifactorial and concerted nature of pathogenicity in A. hydrophila. The large number of putative virulence genes identified in this study will form the basis for further investigation of this emerging pathogen and help to develop effective vaccines, diagnostics, and novel therapeutics.Aeromonas hydrophila is a ubiquitous gram-negative bacterium of aquatic environments, which has been implicated as a causative agent of motile aeromonad septicemia in a variety of aquatic animals (especially freshwater fish species) (2, 37). It causes gastrointestinal and extraintestinal infections in humans, including septicemia, wound infections, and gastroenteritis (16). A number of virulence factors have been identified in A. hydrophila, namely, pili and adhesins (29, 30), O-antigens and capsules (23,48,49), S-layers (10), exotoxins such as hemolysins and enterotoxin (7,14), and a repertoire of exoenzymes which digest cellular components such as proteases, amylases, and lipases (20, 28).The pathogenesis of A. hydrophila is multifactorial. Most studies to date have concentrated on the characterization of a few virulence factors in different animal models by using different strains (10, 34, 42), making it very difficult to evaluate the significance of each gene in the virulence of A. hydrophila. This study aims to identify more putative virulence determinants and characterize them in an integrated manner.Suppressive subtraction hybridization or genomic subtraction offers a genome-level approach to identifying the genetic differences between virulent and avirulent strains of bacteria (22,47). In this study, we used genomic subtraction to identify 22 unique DNA fragments encoding 19 putative virulence factors and seven new open reading frames (ORFs), which are frequently present in a group of virulent strains of A. hydrophila. In...

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A Major Secreted Elastase Is Essential for Pathogenicity of Aeromonas hydrophila

Cited by 139 publications

References 48 publications

Analysis of Carboxy Terminal Domain of Metalloprotease of Elastolytic <i>Aeromonas hydrophila</i>

Analysis of Carboxy Terminal Domain of Metalloprotease of Elastolytic <i>Aeromonas hydrophila</i>

Behavior of an Aeromonas hydrophila aroA Live Vaccine in Water Microcosms

Identification and Characterization of Putative Virulence Genes and Gene Clusters in Aeromonas hydrophila PPD134/91

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